Errors in mitochondrial DNA replication have been hypothesized to drive aging and age-associated disease. The best evidence to date of a causal role for mitochondrial mutation in aging is the finding of an accelerated aging phenotype in mice expressing an error-prone form of the mitochondrial DNA polymerase; however, it is challenging to interpret data from studies performed with proteins having compromised function. Accordingly, I propose to develop a mitochondrial DNA polymerase with enhanced replication fidelity. An anti-mutator mitochondrial polymerase will serve as a model to determine the true role of mitochondrial DNA mutation in health and lifespan. In Specific Aim 1,1 will create specific mutations in the mouse mitochondrial DNA polymerase, utilizing known anti-mutator substitutions that have been characterized in prokaryotic model systems, and determine the effect of these substitutions on polymerase fidelity. In Specific Aim 2,1 will analyze the most promising anti-mutator polymerase mutants by quantitatively determining their activity, error rate, mutation spectrum, and biochemical properties. In Specific Aim 3,1 will determine the in vivo consequences of enhanced fidelity of mitochondrial DNA synthesis by expressing the anti-mutator polymerases in eukaryotic cells. A better understanding of the role of mitochondrial DNA replication fidelity in health and lifespan will lend insight into the fundamental mechanisms underlying aging itself. Because a large number of human diseases increase in both incidence and severity as a function of age, a better understanding of the aging process may result in strategies to delay or prevent the occurrence of disease. [unreadable] [unreadable] [unreadable]